Mechanisms underlying the specificity of aminoacyl-tRNA synthesis will be studied in the glutamyl and glutaminyl systems. First, E. coli glutaminyl-tRNA synthetase will be used as a model system to break new ground in our understanding of intramolecular communication between the amino acid and tRNA binding sites of tRNA synthetases. Alanine scanning mutagenesis will be used to create a mapping of which enzyme-tRNA contacts influence the affinity for glutamine. This overall study will then be followed with an in-depth analysis of the roles of indirect readout, electrostatics, and conformational strain in communication between the tRNA acceptor stem interface and the adjacent glutamine binding site. Next, we will use a novel chemical and enzymatic synthesis approach to study the importance of the 2-thiouridine modification at the anticodon wobble base position, in both the glutaminyl and glutamyl systems. Further studies are proposed on a diverse set of glutamyl- tRNA synthetases, to uncover the structural and energetic basis for relaxation of tRNA specificity in non-discriminating enzymes. Finally, further studies on an engineered hybrid GlnRS enzyme with significant capacity to synthesize glutamylated tRNAGln are proposed. Throughout the application, a common theme is the integration of pre-steady state kinetic measurements with X-ray structural data. A new theme is the further inclusion of in silico bioinformatics approaches to derive correlations from the exhaustive tRNA and tRNA synthetase databases. The elucidation of how induced fit and indirect readout mechanisms control tRNA and amino acid specificities in these systems will be relevant to understanding such processes in more complex particles such as the ribosome. Further, there is great potential for developing novel antimicrobial compounds based on the discrimination between human and bacterial synthetases. Finally, engineering of tRNA synthetases to expand the genetic code may open the door to novel protein-based therapeutics and has potential to impact the development of therapies for many human diseases. PUBLIC HEALTH RELEVANCE: Aminoacyl-tRNA synthetases are essential enzymes in every living cell. Understanding how they function in protein synthesis is crucial to public health because many human diseases can be traced to defective proteins. In addition to this very general role in synthesizing all proteins, one aminoacyl-tRNA synthetase studied in this project is also involved in regulating the expression of genes that control inflammation in humans. This enzyme, the human glutamyl-tRNA synthetase, thus has a particular known relevance to public health.